Rapid advances in recombinant DNA technology has led to the identification and isolation of many novel genes. Consequently, there is a need to express genes in a heterologous cell system to obtain material for structure-function studies, diagnostic reagents, testing, and therapy. Both eukaryotic and prokaryotic systems have been developed for the expression of heterologous genes. Perhaps the best known example of a prokaryotic expression system is Escherichia coli. Using prokaryotic systems to express eukaryotic genes, however, may create difficulties such as improper folding and improper post-translational modifications, which may affect a protein's function, activity, or stability. Moreover, the expressed product may be toxic to the prokaryotic cell or be produced in such large quantities that they interfere with cell growth. Thus, to overcome the drawbacks associated with expressing eukaryotic genes in prokaryotic cells, it may be necessary to express these genes in a eukaryotic cell. Expressing eukaryotic genes in eukaryotic cells provides for high levels of expression, proper post-translational modifications, and proper folding of the expression products. Eukaryotic expression systems may be, for example, mammalian, insect, and yeast cells.
Intrinsic difficulties limit intracellular expression. For example, intracellularly expression products must be purified from the host cell. Furthermore, expression products may sometimes be encapsulated be in inclusion bodies. Intracellular proteases may also degrade intracellularly expressed products. Some expressed products are produced in inactive forms possibly due to improper folding when produced intracellularly. The problems associated with intracellular expression may lead to decreased yield and make purification expensive and time consuming. Thus, it is desirable to use an expression system designed to transfer the heterologous protein from the intracellular environment.
Secretion of the heterologous protein provides an alternative to intracellular expression. Sambrook et al. Molecular Cloning 17.31 (1989) provide examples of expression vectors designed for the secretion of heterologous proteins in E. coli. Generally, secretion of the expressed product is accomplished by operably linking a nucleotide sequence encoding a secretion signal peptide to a nucleotide sequence encoding the heterologous polypeptide. The secretion signal peptide directs the expressed product through the secretory pathway and into the extracellular medium. Secretion of expression products may allow for proper folding and post-translational modification of the expressed product. Secretion of expression products is also advantageous in that purification of the protein of interest does not require harsh treatment to obtain the protein from within the cell. Thus expression systems providing for secretion of the expressed product into the extracellular medium may avoid problems associated with intracellular expression.
Expression of the heterologous protein on the surface of the host cell is also desirable in certain applications. Surface display is accomplished by operably linking a nucleic acid sequence encoding an anchor domain peptide to a nucleotide sequence encoding a heterologous polypeptide that is subsequently shuttled through the secretory pathway and “displayed” on the cell. The anchor domain peptide may be covalently or noncovalently attached to the cell wall. Surface display of heterologous proteins on the host cell allows for post-translational processing, efficient folding, and activity. Surface display is useful for obtaining specific antibodies, determining enzyme specificity, studying protein-protein interactions, fluorescence-activated cell sorting (FACS), and expression cloning. Both prokaryotic and eukaryotic systems are available for expression on the cell. Using prokaryotic systems for surface display of eukaryotic genes, however, shares similar drawbacks to prokaryotic systems for intracellular and secretion expression of eukaryotic genes. For example, prokaryotic cells do not efficiently express functional eukaryotic proteins and lack the ability to introduce post-translational modifications.
Yeast host cell expression systems are useful for recombinant protein expression. As eukaryotes, yeast provide the advantages of proper folding, post-translational modification, and function, such that heterologous proteins are very similar to native proteins from other eukaryotic sources (e.g., mammals, avians, and plants).
S. cerevisiae was the first, and remains one of the most commonly employed yeast expression systems because its genome and physiology have been extensively characterized. P. pastoris is a methylotrophic yeast that has also been developed for heterologous protein expression. Heterologous protein expression using P. pastoris is advantageous relative to S. cerevisiae because it may be cultured at high densities (150 g/L dry cell weight; 500 OD 600 U/mL) where S. cerevisiae produces ethanol at toxic levels. P. pastoris also provides an advantageous heterologous protein production system for production of human pharmaceuticals because it is inexpensive and the cells readily grow in defined medium that is free of undefined ingredients that may be sources of toxins. Additionally, P. pastoris may be cultured in medium with a relatively low pH and methanol, and thus are less likely to become contaminated by other microorganisms.
Because the yeast secrete only a small amount of native protein into its growth medium, yeast expression systems provide a useful alternative to other expression systems. Constructions for secretion of the protein of interest may use the native secretion signal sequence of the protein of interest. In some cases, however, P. pastoris has not been able to efficiently utilize the native signal sequence of the protein of interest to direct secretion. See e.g., Tschopp et al. (5 Bio/Technology 1305-1308 (1987)). Researchers have successfully used the native secretion signal sequence of the heterologous protein, the S. cerevisiae α-factor pre-pro peptide, and the P. pastoris acid phosphatase (PHO1) signal for secretion of expressed heterologous proteins. However, results have been variable. See e.g., Ilgen et al. (Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems 152-153 (Gerd Gellissen, ed., WILEY-VCH Verlag GmbH & Co. KGaA)); Vedvic et al. (5 J. Ind. Microbiol. 197-201 (1991)); Clare et al. (105 Gene 205-212 (1991)).
Researchers have also successfully developed yeast expression systems for displaying heterologous peptides on the cell surface. Boder and Wittrup, (15 Nature Biotechnol. 533-557 (1997)) disclose a yeast expression system using S. cerevisiae. This method uses a S. cerevisiae-derived α-agglutinin adhesion receptor, which consists of the Aga1 and Aga2 subunits. Aga1 is anchored to the cell wall via a β-glucan covalent linkage and Aga2 is linked to Aga1 by disulfide bonds. Yeast express a heterologous polypeptide linked to Aga2, which is displayed on the cell surface. Wang et al. (56 Curr. Microbiol. 352-357 (2008); PIR1), Mergler et al. (63 Appl. Microbiol. Biotechnol. 418-421 (2004); GPI), and Tamino et al. (22 Biotechnol. Prog. 989-993 (2006); Flo1p) each disclose using a S. cerevisiae-derived anchor domain protein for surface display in P. pastoris cells.
With all the purported advantages, heterologous protein expression in yeast is far from optimal. Accordingly, a need still exists for yeast expression systems providing for expression of heterologous proteins wherein the heterologous protein is transferred from the intracellular environment of the host cell to either the growth media or the cell surface.